U.S. patent application number 14/439041 was filed with the patent office on 2015-10-15 for papanicolaou test for ovarian and endometrial cancers.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY. The applicant listed for this patent is THE JOHNS HOPKINS UNIVERSITY. Invention is credited to Chetan Bettegowda, Luis Diaz, Isaac Kinde, Kenneth W. Kinzler, Nickolas Papadopoulos, Bert Vogelstein, Yuxuan Wang.
Application Number | 20150292027 14/439041 |
Document ID | / |
Family ID | 50627940 |
Filed Date | 2015-10-15 |
United States Patent
Application |
20150292027 |
Kind Code |
A1 |
Kinde; Isaac ; et
al. |
October 15, 2015 |
PAPANICOLAOU TEST FOR OVARIAN AND ENDOMETRIAL CANCERS
Abstract
The recently developed liquid-based Papanicolaou (Pap) smear
allows not only cytologic evaluation but also collection of DNA for
detection of HPV, the causative agent of cervical cancer. We tested
these samples to detect somatic mutations present in rare tumor
cells that might accumulate in the cervix once shed from
endometrial and ovarian cancers. A panel of commonly mutated genes
in endometrial and ovarian cancers was assembled and used to
identify mutations in all 46 endometrial or cervical cancer tissue
samples. We were able also able to identify the same mutations in
the DNA from liquid Pap smears in 100% of endometrial cancers (24
of 24) and in 41% of ovarian cancers (9 of 22). We developed a
sequence-based method to query mutations in 12 genes in a single
liquid Pap smear without prior knowledge of the tumor's
genotype.
Inventors: |
Kinde; Isaac; (Beaumont,
CA) ; Kinzler; Kenneth W.; (Baltimore, MD) ;
Vogelstein; Bert; (Baltimore, MD) ; Papadopoulos;
Nickolas; (Towson, MD) ; Diaz; Luis; (Ellicott
City, MD) ; Bettegowda; Chetan; (Perry Hall, MD)
; Wang; Yuxuan; (Baltimore, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE JOHNS HOPKINS UNIVERSITY |
Baltimore |
MD |
US |
|
|
Assignee: |
THE JOHNS HOPKINS
UNIVERSITY
Baltimore
MD
|
Family ID: |
50627940 |
Appl. No.: |
14/439041 |
Filed: |
October 17, 2013 |
PCT Filed: |
October 17, 2013 |
PCT NO: |
PCT/US2013/065342 |
371 Date: |
April 28, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61719942 |
Oct 29, 2012 |
|
|
|
Current U.S.
Class: |
506/2 ; 435/29;
435/6.11; 435/7.1; 435/7.23; 435/7.4; 435/7.92; 506/16; 506/9 |
Current CPC
Class: |
G01N 33/57449 20130101;
C12Q 2600/156 20130101; C12Q 2600/154 20130101; C12Q 2600/16
20130101; C12Q 2600/158 20130101; G01N 33/57442 20130101; C12Q
1/6886 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
[0001] This invention was made using funds from the National Cancer
Institute and the National Institutes of Health. The U.S.
government retains certain rights under the terms of NCI contract
N01-CN-43309 and NIH grants CA129825 and CA43460.
Claims
1. A method comprising: testing a specimen comprising cells or cell
fragments collected from the gynecological tract of a human subject
for a genetic or epigenetic change in one or more genes, mRNAs, or
proteins mutated in endometrial, fallopian tubal, or ovarian
cancer.
2. The method of claim 1 wherein the change is a substitution
mutation.
3. The method of claim 1 wherein the change is a rearrangement.
4. The method of claim 1 wherein the change is a deletion.
5. The method of claim 1 wherein the change is a loss or gain of
methylation.
6. The method of claim 1 wherein the change is determined with
respect to the bulk of the genes, mRNAs, or proteins present in the
specimen.
7. A method comprising: testing a specimen comprising cells or cell
fragments collected from the gynecological tract of a human subject
for one or more mutations in a gene, mRNA, or protein selected from
the group consisting of CTNNB1, EGFR, PI3KCA, PTEN, TP53, BRAF,
KRAS, AKT1, NRAS, PPP2R1A, APC, FBXW7, ARID1A, CDKN2A, MLL2, RFF43,
and FGFR2.
8. The method of claim 7 wherein the step of testing is performed
on at least 3 of said genes, mRNAs, or proteins.
9. The method of claim 7 wherein the step of testing is performed
on at least 5 of said genes, mRNAs, or proteins.
10. The method of claim 7 wherein the step of testing is performed
on at least 7 of said genes, mRNAs, or proteins.
11. The method of claim 7 wherein the step of testing is performed
on at least 9 of said genes, mRNAs, or proteins.
12. The method of claim 7 wherein the step of testing is performed
on at least 11 of said genes, mRNAs, or proteins.
13. The method of claim 7 wherein the step of testing is performed
on at least 12 of said genes, mRNAs, or proteins.
14. The method of claim 7 wherein the step of testing is performed
in a multiplex assay.
15. The method of claim 7 wherein the step of testing is repeated
over time.
16. The method of claim 7 wherein the liquid Pap specimen is
collected after surgical debulking of an ovarian tumor.
17. A kit for testing a panel of genes in Pap specimens for ovarian
or endometrial cancers, the kit comprising a at least 10 probes or
at least 10 primer pairs, wherein each probe or primer comprises at
least 15 nt of complementary sequence to one of the panel of genes,
wherein the panel is cumulatively complementary to at least 10
different genes, wherein the panel is selected from the group
consisting of CTNNB1, EGFR, PI3KCA, PTEN, TP53, BRAF, KRAS, AKT1,
NRAS, PPP2R1A, APC, FBXW7, ARID1A, CDKN2A, MLL2, RFF43, and
FGFR2.
18. The kit of claim 17 which comprises probes and wherein the
probes are attached to a solid support.
19. The kit of claim 17 which comprises primer pairs, wherein the
primer pairs prime synthesis of a nucleic acid of between 240 and
300 bp.
20. The kit of claim 17 which comprises primer pairs, wherein the
primer pairs prime synthesis of a nucleic acid of between 200 and
325 bp.
21. The kit of claim 17 which comprises primer pairs, wherein the
primer pairs prime synthesis of a nucleic acid of between 60 and
1000 bp.
22. The kit of claim 21 wherein at least one primer from each
primer pair is attached to a solid support.
23. The kit of claim 17 wherein the probe or primer comprises at
least 20 nt of complementary sequence to one of the panel of
genes.
24. A solid support comprising at least 10 probes attached thereto,
wherein each probe comprises at least 15 nt of complementary
sequence to one of a panel of genes, wherein the panel is selected
from the group consisting of CTNNB1, EGFR, PI3KCA, PTEN, TP53,
BRAF, KRAS, AKT1, NRAS, PPP2R1A, APC, FBXW7, ARID1A, CDKN2A, MLL2,
RFF43, and FGFR2, wherein the panel is cumulatively complementary
to at least 10 different genes.
25. A solid support comprising at least 10 primers attached
thereto, wherein each primer comprises at least 15 nt of
complementary sequence to one of a panel of genes, wherein the
panel is selected from the group consisting of CTNNB1, EGFR,
PI3KCA, PTEN, TP53, BRAF, KRAS, AKT1, NRAS, PPP2R1A, APC, FBXW7,
ARID1A, CDKN2A, MLL2, RFF43, and FGFR2, wherein the panel is
cumulatively complementary to at least 10 different genes.
26. The method of claim 1 wherein the specimen is a liquid Pap
specimen.
27. The method of claim 7 wherein the specimen is a liquid Pap
specimen.
28. The method of claim 1 wherein the specimen is collected from
the cervix.
29. The method of claim 7 wherein the specimen is collected from
the cervix.
30. The method of claim 7 wherein the step of testing is performed
by increasing the sensitivity of massively parallel sequencing
instruments with an error reduction technique that allows for the
detection of rare mutant alleles in a range of 1 mutant template
among 5,000 to 1,000,000 wild-type templates.
31. The method of claim 7 wherein the step of testing is performed
by increasing the sensitivity of massively parallel sequencing
instruments with an error reduction technique that includes: a.
assignment of a unique identifier (UID) to each template molecule;
b. amplification of each uniquely tagged template molecule to
create UID-families; and c. redundant sequencing of the
amplification products.
Description
TECHNICAL FIELD OF THE INVENTION
[0002] This invention is related to the area of cancer screening.
In particular, it relates to ovarian and endometrial cancers.
BACKGROUND OF THE INVENTION
[0003] Since the introduction of the Papanicolaou test, the
incidence and mortality of cervical cancer in screened populations
has been reduced by more than 75% (1, 2). In contrast, deaths from
ovarian and endometrial cancers have not substantially decreased
during that same time period. As a result, more than 69,000 women
in the U.S. will be diagnosed with ovarian and endometrial cancer
in 2012. Although endometrial cancer is more common than ovarian
cancer, the latter is more lethal. In the U.S., approximately
15,000 and 8,000 women are expected to die each year from ovarian
and endometrial cancers, respectively (Table 1). World-wide, over
200,000 deaths from these tumors are expected this year alone (3,
4).
[0004] In an effort to replicate the success of cervical cancer
screening, several approaches for the early detection of
endometrial and ovarian cancers have been devised. For endometrial
cancers, efforts have focused on cytology and transvaginal
ultrasound (TVS). Cytology can indeed indicate a neoplasm within
the uterus in some cases, albeit with low specificity (5). TVS is a
noninvasive technique to measure the thickness of the endometrium
based on the fact that endometria harboring a cancer are thicker
than normal endometria (6). As with cytology, screening measurement
of the endometrial thickness using TVS lacks sufficient specificity
because benign lesions, such as polyps, can also result in a
thickened endometrium. Accordingly, neither cytology nor TVS
fulfills the requirements for a screening test (5, 7).
[0005] Even greater efforts have been made to develop a screening
test for ovarian cancer, using serum CA-125 levels and TVS. CA-125
is a high molecular weight transmembrane glycoprotein expressed by
coelomic- and Mullerian-derived epithelia that is elevated in a
subset of ovarian cancer patients with early stage disease (8). The
specificity of CA-125 is limited by the fact that it is also
elevated in a variety of benign conditions, such as pelvic
inflammatory disease, endometriosis and ovarian cysts (9). TVS can
visualize the ovary but can only detect large tumors and cannot
definitively distinguish benign from malignant tumors. Several
clinical screening trials using serum CA-125 and TVS have been
conducted but none has shown a survival benefit. In fact, some have
shown an increase in morbidity compared to controls because false
positive tests elicit further evaluation by laparoscopy or
exploratory laparotomy (10-12).
[0006] Accordingly, the U.S. Preventative Services Task Force, the
American Cancer Society, the American Congress of Obstetricians and
Gynecologists, as well as the National Comprehensive Cancer
Network, do not recommend routine screening for endometrial or
ovarian cancers in the general population. In fact, these
organizations warn that "the potential harms outweigh the potential
benefits" (13-16). An exception to this recommendation has been
made for patients with a hereditary predisposition to ovarian
cancer, such as those with germline mutations in a BRCA gene or
those with Lynch syndrome. It is recommended that BRCA mutation
carriers be screened every 6 months with TVS and serum CA-125,
starting at a relatively early age. Screening guidelines for women
with Lynch syndrome include annual endometrial sampling and TVS
beginning between age 30 and 35 (15, 17).
[0007] The mortality associated with undetected gynecologic
malignancies has made the development of an effective screening
tool a high priority. An important observation that inspired the
current study is that asymptomatic women occasionally present with
abnormal glandular cells (AGCs) detected in a cytology specimen as
part of their routine cervical cancer screening procedure. Although
AGCs are associated with premalignant or malignant disease in some
cases (18-22), it is often difficult to distinguish the AGCs
arising from endocervical, endometrial or ovarian cancer from one
another or from more benign conditions. There is a continuing need
in the art to detect these cancers at an earlier stage than done
currently.
SUMMARY OF THE INVENTION
[0008] According to one aspect of the invention a method is
provided for detecting or monitoring endometrial or ovarian cancer.
A liquid Pap smear of a patient is tested for a genetic or
epigenetic change in one or more genes, mRNAs, or proteins mutated
in endometrial or ovarian cancer. Detection of the change indicates
the presence of such a cancer in the patient.
[0009] According to another aspect of the invention a method is
provided for screening for endometrial and ovarian cancers. A
liquid Pap smear is tested for one or more mutations in a gene,
mRNA, or protein selected from the group consisting of CTNNB1,
EGFR, PI3KCA, PTEN, TP53, BRAF, KRAS, AKT1, NRAS, PPP2R1A, APC,
FBXW7, ARID1A, CDKN2A, MLL2, RFF43, and FGFR2. Detection of the
mutation indicates the presence of such a cancer in the
patient.
[0010] Another aspect of the invention is a kit for testing a panel
of genes in Pap smear samples for ovarian or endometrial cancers.
The kit comprises at least 10 probes or at least 10 primer pairs.
Each probe or primer comprises at least 15 nt of complementary
sequence to one of the panel of genes. At least 10 different genes
are interrogated. The panel is selected from the group consisting
of CTNNB1, EGFR, PI3KCA, PTEN, TP53, BRAF, KRAS, AKT1, NRAS,
PPP2R1A, APC, FBXW7, ARID1A, CDKN2A, MLL2, RFF43, and FGFR2.
[0011] Still another aspect of the invention is a solid support
comprising at least 10 attached probes. Each probe comprises at
least 15 nt of complementary sequence to one of a panel of genes,
wherein the panel is selected from the group consisting of CTNNB1,
EGFR, PI3KCA, PTEN, TP53, BRAF, KRAS, AKT1, NRAS, PPP2R1A, APC,
FBXW7, ARID1A, CDKN2A, MLL2, RFF43, and FGFR2.
[0012] Another aspect of the invention is a solid support
comprising at least 10 primers attached thereto. Each primer
comprises at least 15 nt of complementary sequence to one of a
panel of genes. The panel is selected from the group consisting of
CTNNB1, EGFR, PI3KCA, PTEN, TP53, BRAF, KRAS, AKT1, NRAS, PPP2R1A,
APC, FBXW7, ARID1A, CDKN2A, MLL2, RFF43, and FGFR2.
[0013] These and other embodiments which will be apparent to those
of skill in the art upon reading the specification provide the art
with methods for assessing ovarian and endometrial cancers in a
screening environment using samples that are already routinely
collected.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1. Schematic demonstrating the principle steps of the
procedure described in this study. Tumors cells shed from ovarian
or endometrial cancers are carried into the endocervical canal.
These cells can be captured by the brush used for performing a
routine Pap smear. The brush contents are transferred into a liquid
fixative, from which DNA is isolated. Using next-generation
sequencing, this DNA is queried for mutations that indicate the
presence of a malignancy in the female reproductive tract.
[0015] FIG. 2. Diagram of the assay used to simultaneously detect
mutations in 12 different genes. A modification of the Safe-SeqS
(Safe-Sequencing System) protocol, for simultaneous interrogation
of multiple mutations in a single sample, is depicted. In the
standard Safe-SeqS procedure, only one amplicon is assessed, while
the new system is used to assess multiple amplicons from multiple
genes at once.
[0016] FIG. 3. Mutant allele fractions in Pap smear fluids. The
fraction of mutant alleles from each of 46 pap smear fluids is
depicted. The stage of each tumor is listed on the Y-axis. The
X-axis demonstrates the % mutant allele fraction as determined by
Safe-SeqS.
[0017] FIG. 4. Heat map depicting the results of multiplex testing
of 12 genes in Pap smear fluids. Each block on the y-axis
represents a 30-bp block of sequence from the indicated gene. The
28 samples assessed (14 from women with cancer, 14 from normal
women without cancer) are indicated on the x-axis. Mutations are
indicated as colored blocks, with white indicating no mutation,
yellow indicating a mutant fraction of 0.1% to 1%, orange indicate
a mutant fraction of 1% to 10%, and red indicating a mutant
fraction of >10%.
[0018] FIG. 5. Table 1. Epidemiology of Ovarian and Endometrial
Tumors. The estimated numbers of new cases and deaths in the U.S.
from the major subtypes of ovarian and endometrial cancers are
listed.
[0019] FIG. 6. Table 2. Genetic Characteristics of Ovarian and
Endometrial Cancers. The frequencies of the commonly mutated genes
in ovarian and endometrial cancers are listed.
[0020] FIG. 7. Table S1. Endometrial Cancers (Endometrioid Subtype)
Studied by Whole-exome Sequencing. The summary characteristics of
the 22 cancers used for exome sequencing are listed.
[0021] FIG. 8. Table S3. Mutations Assessed in Pap Smears. Clinical
characteristics of the 46 tumor samples are listed, along with the
mutation identified in each case and the fraction of mutant alleles
identified in the Pap smears.
[0022] FIG. 9. Table S4. Primers Used to Assess Individual
Mutations in Pap Smears. The sequences of the forward and reverse
primers used to test each mutation via Safe-SeqS are listed in
pairs (SEQ ID NO: 4-99, respectively).
[0023] FIG. 10. Table S5. Primers Used to Simultaneously Assess 12
Genes in Pap Smears. The sequences of the forward and reverse
primers for each tested region are listed in pairs (SEQ ID NO:
100-191, respectively).
[0024] FIG. 11. Table S6. Mutations Identified in Pap Smears
through Simultaneous Assessment of 12 Genes. The fraction of mutant
alleles identified in the Pap smears using this approach is listed,
along with the precise mutations identified.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The inventors have developed a test for detecting different
cancers using samples that are already routinely collected for
diagnosing uterine cancer and HPV (human papilloma virus)
infection. Using a panel of genes, a high level of detection of
both endometrial and ovarian cancers was achieved.
[0026] Certain genes have been identified as mutated in a high
proportion of endometrial and ovarian cancers. These include
CTNNB1, EGFR, PI3KCA, PTEN, TP53, BRAF, KRAS, AKT1, NRAS, PPP2R1A,
APC, FBXW7, ARID1A, CDKN2A, MLL2, RFF43, and FGFR2. The test can be
performed on at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, or 17 of these genes. In addition, other genes can be added or
substituted into the panel to achieve a higher rate of
detection.
[0027] Testing for a mutation may be done by analysis of nucleic
acids, such as DNA or mRNA or cDNA. The nucleic acid analytes are
isolated from cells or cell fragments found in the liquid PAP smear
sample. Suitable tests may include any hybridization or sequencing
based assay. Analysis may also be performed on protein encoded by
the genes in the panel. Any suitable test may be used including but
not limited to mass spectrometry. Other suitable assays may include
immunological assays, such as, immunoblotting, immunocytochemistry,
immunoprecipitation, enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), immunoradiometric assays (IRMA) and
immunoenzymatic assays (IEMA), including sandwich assays using
monoclonal or polyclonal antibodies.
[0028] Genetic changes which can be detected are typically
mutations such as deletions, insertions, duplications,
substitutions (missense or nonsense mutations), rearrangements,
etc. Such mutations can be detected inter alia by comparing to a
wild type in another (non-tumor) tissue or fluid of an individual
or by comparing to reference sequences, for example in databases.
Mutations that are found in all tissues of an individual are
germline mutations, whereas those that occur only in a single
tissue are somatic mutations. Epigenetic changes can also be
detected. These may be loss or gain of methylation at specific
locations in specific genes, as well as histone modifications,
including acetylation, ubiquitylation, phosphorylation and
sumoylation.
[0029] Tests may be done in a multiplex format, in which a single
reaction pot is used to detect multiple analytes. Examples of such
tests include amplifications using multiple primer sets,
amplifications using universal primers, array based hybridization
or amplification, emulsion based amplification.
[0030] While probes and primers may be designed to interrogate
particular mutations or particular portions of a gene, mRNA, or
cDNA, these may not be separate entities. For example, probes and
primers may be linked together to form a concatamer, or they may be
linked to one or more solid supports, such as a bead or an
array.
[0031] Kits for use in the disclosed methods may include a carrier
for the various components. The carrier can be a container or
support, in the form of, e.g., bag, box, tube, rack, and is
optionally compartmentalized. The kit also includes various
components useful in detecting mutations, using the above-discussed
detection techniques. For example, the detection kit may include
one or more oligonucleotides useful as primers for amplifying all
or a portion of the target nucleic acids. The detection kit may
also include one or more oligonucleotide probes for hybridization
to the target nucleic acids. Optionally the oligonucleotides are
affixed to a solid support, e.g., incorporated in a microarray
included in the kit or supplied separately.
[0032] Solid supports may contain one single primer or probe or
antibody for detecting a single gene, protein, mRNA, or portion of
a gene. A solid support may contain multiple primers, probes, or
antibodies. They may be provided as a group which will interrogate
mutations at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
or 17 of the genes of the desired panel. The panel may be selected
from or comprise CTNNB1, EGFR, PI3KCA, PTEN, TP53, BRAF, KRAS,
AKT1, NRAS, PPP2R1A, APC, FBXW7, ARID1A, CDKN2A, MLL2, RFF43, and
FGFR2.
[0033] Primer pairs may be used to synthesize amplicons of various
sizes. Amplicons may be for example from 50, 60, 75, 100, 125, 150,
200, 140, 180 bp in length. Amplicons may run up to 200, 250, 300,
400, 500, 750, 1000 bp in length, as examples. The size of the
amplicon may be limited by the size and/or quality of the template
retrieved from the liquid PAP smear. Probes and primers for use in
the invention may contain a wild-type sequence or may contain a
sequence of a particular mutant.
[0034] In one embodiment, the test can be performed using samples
that are collected over time. The test results can be compared for
quantitative or qualitative changes. Such analysis can be used
after a potentially curative therapy, such as surgery.
[0035] Georgios Papanicolaou published his seminal work, entitled
"Diagnosis of Uterine Cancer by the Vaginal Smear," in 1943 (31).
At that time, he suggested that endocervical sampling could, in
theory, be used to detect not only cervical cancers but also other
cancers arising in the female reproductive tract, including
endometrial carcinomas. The research reported here moves us much
closer to that goal. In honor of Papanicolaou's pioneering
contribution to the field of early cancer detection, we have named
the approach described herein as the "PapGene" test.
[0036] One of the most important developments over the last several
years is the recognition that all human cancers are the result of
mutations in a limited set of genes and an even more limited set of
pathways through which these genes act (32). The whole-exome
sequencing data we present, combined with previous genome-wide
studies, provide a striking example of the common genetic features
of cancer (FIG. 6, Table 2). Through the analysis of particular
regions of only 12 genes (FIG. 11, table S5), we could detect at
least one driver mutation in the vast majority of nine different
gynecologic cancers (FIG. 5, Table 1). Though several of these 12
genes were tumor suppressors, and therefore difficult to
therapeutically target, knowledge of their mutational patterns
provides unprecedented opportunities for cancer diagnostics.
[0037] The most important finding in this paper is that significant
amounts of cells or cell fragments from endometrial and ovarian
cancers are present in the cervix and can be detected through
molecular genetic approaches. Detection of malignant cells from
endometrial and ovarian carcinomas in cervical cytology specimens
is relatively uncommon. Microscopic examination cannot always
distinguish them from one another, from cervical carcinomas, or
from more benign conditions. Our study showed that 100% of
endometrial cancers (n=24), even those of low grade, and 41% of
ovarian cancers (n=22), shed cells into the cervix that could be
detected from specimens collected as part of routine Pap smears.
This finding, in conjunction with technical advances allowing the
reliable detection of mutations present in only a very small
fraction of DNA templates, provided the foundation for the PapGene
test.
[0038] This study demonstrates the value of sensitive endocervical
DNA testing but there are many issues that need to be addressed
before optimal clinical use is achieved. The test, even in its
current format, appears to be promising for screening endometrial
cancer, as the data in FIG. 3 show that even the lowest stage
endometrial cancers could be detected through the analysis of DNA
in Pap smear fluid through Safe-SeqS. However, only 41% of ovarian
cancers could be detected in Pap smears even when the mutations in
their tumors were known. In eight of the nine Pap smears from
ovarian cancer patients that contained detectable mutations, the
mutant allele fractions were >0.1% and therefore within the
range currently detectable by PapGene testing (FIG. 9, table S3).
Further improvements in the technology could increase the technical
sensitivity of the PapGene test and allow it to detect more ovarian
cancers. Other strategies to increase this sensitivity involve
physical maneuvers, such as massaging the adnexal region during the
pelvic examination or by performing the PapGene test at specified
times during the menstrual cycle. Development of an improved method
of collection may also be able to improve sensitivity. The current
liquid specimen is designed for the detection of cervical cancer
and as such utilizes a brush that collects cells from the
ectocervix and only minimally penetrates the endocervical canal. A
small cannula that can be introduced into the endometrial cavity
similar to the pipelle endometrial biopsy instrument could
theoretically obtain a more enriched sample of cells coming from
the endometrium, fallopian tube and ovary (33).
[0039] The high sensitivity and the quantitative nature of the
PapGene test also opens the possibility of utilizing it to monitor
the response to hormonal agents (e.g., progestins) used to treat
young women with low risk endometrial cancers. Some of these women
choose to preserve fertility, undergoing medical therapy rather
than hysterectomy (34). The detection of pre-symptomatic ovarian
cancers, even if advanced, could also be of benefit. Although not
entirely analogous, it has been demonstrated that one of the most
important prognostic indicators for ovarian cancer is the amount of
residual disease after surgical debulking Initially, debulking was
considered optimal if the residual tumor was less than 2 cm.
Subsequently, the threshold was reduced to 1 cm and surgeons now
attempt to remove any visible tumor. With each improvement in
surgical debulking, survival has lengthened (35). A small volume of
tumor is likely to be more sensitive to cytotoxic chemotherapy than
the large, bulky disease typical of symptomatic high-grade serous
carcinoma.
[0040] An essential aspect of the screening approach described here
is that it should be relatively inexpensive and easily incorporated
into the pelvic examination. Evaluation of HPV DNA is already part
of routine Pap smear testing because HPV analysis increases the
test's sensitivity (36, 37). The DNA purification component of the
PapGene test is identical to that used for HPV, so this component
is clearly feasible. The preparation of DNA, multiplex
amplification, and the retail cost of the sequencing component of
the PapGene test can also be performed at a cost comparable to a
routine HPV test in the U.S. today. Note that the increased
sensitivity provided by the Safe-SeqS component of the PapGene test
(see Example 6) can be implemented on any massively parallel
sequencing instrument, not just those manufactured by Illumina.
With the reduction in the cost of massively parallel sequencing
expected in the future, PapGene testing should become even less
expensive.
[0041] There are millions of Pap smear tests performed annually in
the U.S. Could PapGene testing be performed on such a large number
of specimens? We believe so, because the entire DNA purification
and amplification process can be automated, just as it is for HPV
testing. Though it may now seem unrealistic to have millions of
these sophisticated sequence-based tests performed every year, it
would undoubtedly have seemed unrealistic to have widespread,
conventional Pap smear testing performed when Papanicolaou
published his original paper (31). Even today, when many cervical
cytology specimens are screened using automated technologies, a
significant percentage require evaluation by a skilled
cytopathologist. In contrast, the analysis of PapGene testing is
done completely in silico and the read-out of the test is objective
and quantitative.
[0042] In sum, PapGene testing has the capacity to increase the
utility of conventional cytology screening through the unambiguous
detection of endometrial and ovarian carcinomas. In addition to the
analysis of much larger numbers of patients with and without
various types of endometrial, ovarian, and fallopian tube cancers,
the next step in this line of research is to include genes altered
in cervical cancer as well as HPV amplicons in the multiplexed
Safe-SeqS assay (FIG. 11, table S5). These additions will provide
information that could be valuable for the management of patients
with the early stages of cervical neoplasia, as HPV positivity
alone is not specific for the detection of cervical cancer and its
precursor lesions, particularly in young, sexually active women who
frequently harbor HPV infections in the absence of neoplasia.
[0043] The above disclosure generally describes the present
invention. All references disclosed herein are expressly
incorporated by reference. A more complete understanding can be
obtained by reference to the following specific examples which are
provided herein for purposes of illustration only, and are not
intended to limit the scope of the invention.
Example 1
[0044] We reasoned that more sophisticated molecular methods might
be able to detect the presence of cancer cells in endocervical
specimens at higher sensitivities and specificities than possible
with conventional methods. In particular, we hypothesized that
somatic mutations characteristic of endometrial and ovarian cancers
would be found in the DNA purified from routine liquid-based Pap
smears (henceforth denoted as "Pap smears"; FIG. 1). Unlike
cytologically abnormal cells, such oncogenic DNA mutations are
specific, clonal markers of neoplasia that should be absent in
non-neoplastic cells. However, we did not know if such DNA would
indeed be present in endocervical specimens, and we did not know if
they would be present in a sufficient amount to detect them. The
experiments described here were carried out to test our
hypothesis.
[0045] There were four components to this study: I. Determination
of the somatic mutations typically present in endometrial and
ovarian cancers; II. Identification of at least one mutation in the
tumors of 46 patients with these cancers; III. Determination of
whether the mutations identified in these tumors could also be
detected in Pap smears from the same patients; and IV. Development
of a technology that could directly assess cells from Pap smears
for mutations commonly found in endometrial or ovarian cancers.
Example 2
Prevalence of Somatically Mutated Genes in Endometrial and Ovarian
Cancers
[0046] There are five major histopathologic subtypes of ovarian
cancers. The most prevalent subtype is high grade serous (60% of
total), followed by endometrioid (15%), clear cell (10%), and
low-grade serous carcinoma (8%) (Table 1). Genome-wide studies have
identified the most commonly mutated genes among the most prevalent
ovarian cancer subtypes (Table 2) (23-25).
[0047] Such comprehensive studies have not yet been reported for
the endometrioid and mucinous subtypes, collectively representing
.about.20% of ovarian cancer cases (Table 1). However, commonly
mutated genes in the endometrioid and mucinous subtypes have been
reported (26). In aggregate, the most commonly mutated gene in
epithelial ovarian cancers was TP53, which was mutated in 69% of
these cancers (Table 2). Other highly mutated genes included
ARID1A, BRAF, CTNNB1, KRAS, PIK3CA, and PPP2R1A (Table 2).
[0048] Among endometrial cancers, the endometrioid subtype is by
far the most common, representing 85% of the total (Table 1).
Because cancers of this subtype are so frequent and have not been
analyzed at a genome-wide level, we evaluated them through
whole-exome sequencing. The DNA purified from 22 sporadic
endometrioid carcinomas, as well as from matched non-neoplastic
tissues, was used to generate 44 libraries suitable for massively
parallel sequencing. The clinical aspects of the patients and
histopathologic features of the tumors are listed in table S1.
Though the examination of 22 cancers cannot provide a comprehensive
genome landscape of a tumor type, it is adequate for diagnostic
purposes--as these only require the identification of the most
frequently mutated genes.
[0049] Among the 44 libraries, the average coverage of each base in
the targeted region was 149.1 with 88.4% of targeted bases
represented by at least ten reads. Using stringent criteria for the
identification of somatic mutations (as described in Materials and
Methods), the sequencing data clearly demarcated the tumors into
two groups: ten cancers (termed the N Group, for non-highly
mutated) harbored <100 somatic mutations per tumor (median 32,
range 7 to 50), while 12 cancers (termed the H Group, for highly
mutated) harbored >100 somatic mutations per tumor (median 674,
range 164 to 4,629) (FIG. 7, table S1).
[0050] The high number of mutations in the Group H tumors was
consistent with a deficiency in DNA repair. Eight of the 12 Group H
tumors had microsatellite instability (MSI-H, table S1), supporting
this conjecture. Moreover, six of the Group H tumors contained
somatic mutations in the mismatch repair genes MSH2 or MSH6, while
none of the Group N cancers contained mutations in mismatch repair
genes. Mismatch repair deficiency is known to be common among
endometrial cancers and these tumors occur in 19-71% of women with
inherited mutations of mismatch repair genes (i.e., patients with
the Hereditary Nonpolyposis Colorectal Cancer) (27).
[0051] 12,795 somatic mutations were identified in the 22 cancers.
The most commonly mutated genes included the PIK3 pathway genes
PTEN and PIK3CA (28), the APC pathway genes APC and CTNNB1, the
fibroblast growth factor receptor FGFR2, the adapter protein FBXW7,
and the chromatin-modifying genes ARID1A and MLL2 (Table 2). Genes
in these pathways were mutated in both Group N and H tumors. Our
results are consistent with prior studies of endometrioid
endometrial cancer that had evaluated small numbers of genes,
though mutations in FBXW7, MLL2 and APC had not been appreciated to
occur as frequently as we found them. It was also interesting that
few TP53 mutations (5%) were found in these endometrial cancers
(Table 2), a finding also consistent with prior studies.
[0052] Papillary serous carcinomas of the endometrium account for
10-15% of endometrial cancers, and a recent genome-wide sequencing
study of this tumor subtype has been published (29). The most
common mutations in this subtype are listed in Table 2. The least
common subtype of endometrial cancers is clear cell carcinomas,
which occur in <5%. Genes reported to be mutated in these
cancers were garnered from the literature (Table 2).
Example 3
Identification of Mutations in Tumor Tissues
[0053] We acquired tumors from 46 cancer patients in whom Pap
smears were available. These included 24 patients with endometrial
cancers and 22 with ovarian cancers; clinical and histopathologic
features are listed in table S3.
[0054] Somatic mutations in the 46 tumors were identified through
whole-exome sequencing as described above or through targeted
sequencing of genes frequently mutated in the most common subtypes
of ovarian or endometrial cancer (Table 2). Enrichment for these
genes was achieved using a custom solid phase capture assay
comprised of oligonucleotides ("capture probes") complementary to a
panel of gene regions of interest. For the oncogenes, we only
targeted their commonly mutated exons, whereas we targeted the
entire coding regions of the tumor suppressor genes.
[0055] Illumina DNA sequencing libraries were generated from tumors
and their matched non-neoplastic tissues, then captured with the
assay described above. Following amplification by PCR, four to
eight captured DNA libraries were sequenced per lane on an Illumina
GA IIx instrument. In each of the 46 cases, we identified at least
one somatic mutation (table S3) that was confirmed by an
independent assay, as described below.
Example 4
Identification of Somatic Mutations in Pap Smears
[0056] In the liquid-based Pap smear technique in routine use
today, the clinician inserts a small brush into the endocervical
canal during a pelvic exam and rotates the brush so that it
dislodges and adheres to loosely attached cells or cell fragments.
The brush is then placed in a vial of fixative solution (e.g.,
ThinPrep). Some of the liquid from the vial is used to prepare a
slide for cytological analysis or for purification of HPV DNA. In
our study, an aliquot of the DNA purified from the liquid was used
to assess for the presence of DNA from the cancers of the 46
patients described above. Preliminary studies showed that the fixed
cells or cell fragments in the liquid, pelleted by centrifugation
at 1,000 g for five minutes, contained >95% of the total DNA in
the vial. We therefore purified DNA from the cell pellets when the
amount of available liquid was greater than 3 mL (as occurs with
some liquid-based Pap smear kits) and, for convenience, purified
DNA from both the liquid and cells when smaller amounts of liquid
were in the kit. In all cases, the purified DNA was of relatively
high molecular weight (95%>5 kb). The average amount of DNA
recovered from the 46 Pap smears was 49.3.+-.74.4 ng/ml (table
S3).
[0057] We anticipated that, if present at all, the amount of DNA
derived from neoplastic cells in the Pap smear fluid would be
relatively small compared to the DNA derived from normal cells
brushed from the endocervical canal. This necessitated the use of
an analytic technique that could reliably identify a rare
population of mutant alleles among a great excess of wild-type
alleles. A modification of one of the Safe-SeqS (Safe-Sequencing
System) procedures described in (30) was designed for this purpose
(FIG. 2).
[0058] In brief, a limited number of PCR cycles was performed with
a set of gene-specific primers. One of the primers contained 14
degenerate N bases (equal probability of being an A, C, G, or T)
located 5' to its gene-specific sequence, and both primers
contained sequences that permitted universal amplification in the
next step. The 14 N's formed unique identifiers (UID) for each
original template molecule. Subsequent PCR products generated with
universal primers were purified and sequenced on an Illumina MiSeq
instrument. If a mutation preexisted in a template molecule, that
mutation should be present in every daughter molecule containing
that UID, and such mutations are called "supermutants" (30).
Mutations not occurring in the original templates, such as those
occurring during the amplification steps or through errors in base
calling, should not give rise to supermutants. The Safe-SeqS
approach used here is capable of detecting 1 mutant template among
5,000 to 1,000,000 wild-type templates, depending on the amplicon
and the position within the amplicon that is queried (30).
[0059] We designed Safe-SeqS primers (table S4) to detect at least
one mutation from each of the 46 patients described in table S3. In
the 24 Pap smears from patients with endometrial cancers, the
mutation present in the tumor was identified in every case (100%).
The median fraction of mutant alleles was 2.7%, and ranged from
0.01% to 78% (FIG. 3 and table S3). Amplifications of DNA from
non-neoplastic tissues were used as negative controls in these
experiments to define the detection limits of each queried
mutation. In all cases, the fraction of mutant alleles was
significantly different from the background mutation levels
determined from the negative controls (P<0.001, binomial test).
There was no obvious correlation between the fraction of mutant
alleles and the histopathologic subtype or the stage of the cancer
(FIG. 3 and table S3).
[0060] In two endometrial cancer cases, two mutations found in the
tumor DNA were evaluated in the Pap smears (table S3). In both
cases, the mutations were identified in DNA from the Pap smear
(table S3). Moreover, the ratios between the mutant allele
fractions of the two mutations in the Pap smears were correlated
with those of the corresponding tumor samples. For example, in the
Pap smear of case PAP 083 the mutant allele fractions for the
CTNNB1 and PIK3CA mutations were 0.143% and 0.064%, respectively--a
ratio of 2.2 (=0.14% to 0.064%). In the primary tumor from PAP 083,
the corresponding ratio was 2.0 (79.5% to 39.5%).
[0061] Similar analysis of Pap smear DNA from ovarian cancer
patients revealed detectable mutations in nine of the 22 patients
(41%). The fraction of mutant alleles was smaller than in
endometrial cancers (median of 0.49%, range 0.021% to 5.9%; see
FIG. 3 and table S3). All but one of the cases with detectable
mutations were epithelial tumors; the exception was a dysgerminoma,
a malignant germ cell tumor of the ovary (table S3). As with
endometrial cancers, there was no statistically significant
correlation between the fraction of mutant alleles and
histopathologic criteria. However, most ovarian cancers are
detected only at an advanced stage, and this was reflected in the
patients available in our cohort.
Example 5
A Genetic Test for Screening Purposes
[0062] The results described above document that mutant DNA
molecules from most endometrial cancers and some ovarian cancers
can be found in routinely collected Pap smears. However, in all 46
cases depicted in FIG. 3, a specific mutation was known to occur in
the tumor, and an assay was subsequently designed to determine
whether that mutation was also present in the corresponding Pap
smears. In a screening setting, there obviously would be no known
tumor prior to the test. We therefore designed a prototype test
based on Safe-SeqS that could be used in a screening setting (FIG.
2).
[0063] This multiplexed approach included 50 primer pairs that
amplified segments of 241 to 296 bp containing frequently mutated
regions of DNA. The regions to be amplified were chosen from the
results described in Section I and included exons from APC, AKT1,
BRAF, CTNNB1, EGFR, FBXW7, KRAS, PIK3CA, PPP2R1A, PTEN, and TP53.
In control experiments, 46 of the 50 amplicons were shown to
provide information on a minimum of 2,500 templates; the number of
templates sequenced can be determined directly from SafeSeqS-based
sequencing (FIG. 2). Given the accuracy of SafeSeqS, this number
was adequate to comfortably detect mutations existing in >0.1%
of template molecules (30). The regions covered by these 46
amplicons (table S5), encompassing 10,257 bp, were predicted to be
able to detect at least one mutation in >90% of either
endometrial or ovarian cancers.
[0064] This test was applied to Pap smears of 14 cases--twelve
endometrial and two ovarian--as well as 14 Pap smears collected
from normal women. The 14 cancer cases were arbitrarily chosen from
those which had mutant allele fractions >0.1% (table S3) and
therefore above the detection limit of the multiplexed assay. In
all 14 Pap smears from women with cancer, the mutation expected to
be present (table S3) was identified (FIG. 4 and table S6). The
fraction of mutant alleles in the multiplexed test was similar to
that observed in the original analysis of the same samples using
only one Safe-SeqS primer pair per amplicon (table S3 and table
S6). Importantly, no mutations were detected in the 14 Pap smears
from women without cancer (FIG. 4; see Materials and Methods).
Example 6
Materials and Methods
Patient Samples
[0065] All samples for this study were obtained using protocols
approved by the Institutional Review Boards of The Johns Hopkins
Medical Institutions (Baltimore, Md.), Memorial Sloan Kettering
Cancer Center (New York, N.Y.), University of Sao Paulo (Sao Paulo,
Brazil), and ILSbio, LLC (Chestertown, Md.). Demographic, clinical
and pathologic staging data was collected for each case. All
histopathology was centrally re-reviewed by board-certified
pathologists. Staging was based on 2009 FIGO criteria (38).
[0066] Fresh-frozen tissue specimens of surgically resected
neoplasms of the ovary and endometrium were analyzed by frozen
section to assess neoplastic cellularity by a board-certified
pathologist. Serial frozen sections were used to guide the trimming
of Optimal Cutting Temperature (OCT) compound embedded frozen
tissue blocks to enrich the fraction of neoplastic cells for DNA
extraction.
[0067] Formalin-fixed paraffin embedded (FFPE) tissue samples were
assessed by a board-certified pathologist (Propath LLC, Dallas,
Tex.) for tumor cellularity and to demarcate area of high tumor
cellularity. Tumor tissue from serial 10 micron sections on slides
from the original tumor block were macrodissected with a razorblade
to enrich the fraction of neoplastic cells for DNA extraction.
[0068] The source of normal DNA was matched whole blood or
non-neoplastic normal adjacent tissue.
[0069] Liquid-based Pap smears were collected using cervical
brushes and transport medium from Digene HC2 DNA Collection Device
(Qiagen) or ThinPrep 2000 System (Hologic) and stored using the
manufacturer's recommendations.
[0070] Unless otherwise indicated, all patient-related values are
reported as mean.+-.1 standard deviation.
DNA Extraction
[0071] DNA was purified from tumor and normal tissue as well as
liquid-based Pap Smears using an AllPrep kit (Qiagen) according to
the manufacturer's instructions. DNA was purified from tumor tissue
by adding 3 mL RLTM buffer (Qiagen) and then binding to an AllPrep
DNA column (Qiagen) following the manufacturer's protocol. DNA was
purified from Pap smear liquids by adding five volumes of RLTM
buffer when the amount of liquid was less than 3 mL. When the
amount of liquid was >3 mL, the cells and cell fragments were
pelleted at 1,000.times.g for five minutes and the pellets were
dissolved in 3 mL RLTM buffer. DNA was quantified in all cases with
qPCR, employing the primers and conditions previously described
(39).
Microsatellite Instability Testing
[0072] Microsatellite instability was detected using the MSI
Analysis System (Promega), containing five mononucleotide repeats
(BAT-25, BAT-26, NR-21, NR-24 and MONO-27) and two pentanucleotide
repeat loci, per the manufacturer's instructions. Following
amplification, the fluorescent PCR products were sized on an
Applied Biosystems 3130 capillary electrophoresis instrument
(Invitrogen). Tumor samples were designated as follows: MSI-high if
two or more mononucleotides varied in length compared to the
germline DNA; MSI-low if only one locus varied; and microsatellite
stable (MSS) if there was no variation compared to the germline.
Pentanucleotide loci confirmed identity in all cases.
Preparation of Illumina DNA Libraries and Capture for Exomic
Sequencing
[0073] Preparation of Illumina genomic DNA libraries for exomic and
targeted DNA captures was performed according to the manufacturer's
recommendations. Briefly, 1-3 .mu.g of genomic DNA was used for
library preparation using the TruSeqDNA Sample Preparation Kit
(Illumina). The DNA was acoustically sheared (Covaris) to a target
size of .about.200 bp. The fragments were subsequently end-repaired
to convert overhangs into blunt ends. A single "A" nucleotide was
then added to the 3' ends of blunt fragments to prevent them from
later self-ligation; a corresponding "T" on the 3' end of adaptor
molecules provided the complementary overhang. Following ligation
to adaptors, the library was amplified with 8-14 cycles of PCR to
ensure yields of 0.5 and 4 .mu.g for exomic and targeted gene
captures, respectively.
[0074] Exomic capture was performed with the SureSelect Human Exome
Kit V 4.0 (Agilent) according to the manufacturer's protocol, with
the addition of TruSeq index-specific blocks in the hybridization
mixture
(AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-XXXXXX-ATCTCGTATGCCGTCTTCTGCTTGT
(SEQ ID NO: 1), where the six base pair "XXXXXX" denotes one of 12
sample-specific indexes).
Targeted Gene Enrichment
[0075] Targeted gene enrichment was performed by modifications of
previously described methods (40, 41). In brief, targeted regions
of selected oncogenes and tumor suppressor genes were synthesized
as oligonucleotide probes by Agilent Technologies. Probes of 36
bases were designed to capture both the plus and the minus strand
of the DNA and had a 33-base overlap. The oligonucleotides were
cleaved from the chip by incubating with 3 mL of 35% ammonium
hydroxide at room temperate for five hours. The solution was
transferred to two 2-ml tubes, dried under vacuum, and redissolved
in 400 .mu.L of ribonuclease (RNase)- and deoxyribonuclease
(DNase)-free water. Five microliters of the solution was used for
PCR amplification with primers complementary to the 12-base
sequence common to all probes: 5'-TGATCCCGCGACGA*C-3' (SEQ ID NO:
2) and 5'-GACCGCGACTCCAG*C-3' (SEQ ID NO: 3), with * indicating a
phosphorothioate bond. The PCR products were purified with a
MinElute Purification Column (Qiagen), end-repaired with End-IT DNA
End-Repair Kit (Epicentre), and then purified with a MinElute
Purification Column (Qiagen). The PCR products were ligated to form
concatamers as described (40).
[0076] The major difference between the protocol described in (40,
41) and the one used in the present study involved the
amplification of the ligated PCR products and the solid phase
capture method. The modifications were as follows: 50 ng of ligated
PCR product was amplified using the REPLI-g Midi Kit (Qiagen) with
the addition of 2.5 nmol Biotin-dUTP (Roche) in a 27.5 .mu.L
reaction. The reaction was incubated at 30.degree. C. for 16 hours,
the polymerase was inactivated at 65.degree. C. for 3 mins. The
amplified probes were purified with QiaQuick PCR Purification
Columns (Qiagen). For capture, 4-5 .mu.g of library DNA was
incubated with 1 .mu.g of the prepared probes in a hybridization
mixture as previously described (40). The biotinylated probes and
captured library sequences were subsequently purified using 500
.mu.g Dynabeads.RTM. MyOne Streptavidin (Invitrogen). After washing
as per the manufacturer's recommendations, the captured sequences
were eluted with 0.1 M NaOH and then neutralized with 1M Tris-HCl
(pH 7.5). Neutralized DNA was desalted and concentrated using a
QIAquick MinElute Column (Qiagen) in 20 .mu.L. The elute was
amplified in a 100 .mu.L Phusion Hot Start II (Thermo Scientific)
reaction containing 1.times. Phusion HF buffer, 0.25 mM dNTPs, 0.5
.mu.M each forward and reverse TruSeq primers, and 2 U polymerase
with the following cycling conditions: 98.degree. C. for 30 s; 14
cycles of 98.degree. C. for 10 s, 60.degree. C. for 30 s,
72.degree. C. for 30 s; and 72.degree. C. for 5 min. The amplified
pool containing enriched target sequences was purified using an
Agencourt AMPure XP system (Beckman) and quantified using a 2100
Bioanalyzer (Agilent).
Next-Generation Sequencing and Somatic Mutation Identification
[0077] After capture of targeted sequences, paired-end sequencing
using an Illumina GA IIx Genome Analyzer provided 2.times.75 base
reads from each fragment. The sequence tags that passed filtering
were aligned to the human genome reference sequence (hg18) and
subsequent variant-calling analysis was performed using the ELANDv2
algorithm in the CASAVA 1.6 software (Illumina). Known
polymorphisms recorded in dbSNP were removed from the analysis.
Identification of high confidence mutations was performed as
described previously (24).
Assessment of Low-Frequency Mutations
[0078] Primer Design. We attempted to design primer pairs to detect
mutations in the 46 cancers described in the text. Primers were
designed as described (30), using Primer3. (42) Sixty percent of
the primers amplified the expected fragments; in the other 40%, a
second or third set of primers had to be designed to reduce primer
dimers or non-specific amplification.
[0079] Sequencing Library Preparation. Templates were amplified as
described previously (30), with modifications that will be
described in full elsewhere. In brief, each strand of each template
molecule was encoded with a 14 base unique identifier
(UID)--comprised of degenerate N bases (equal probability of being
an A, C, G, or T)--using two to four cycles of amplicon-specific
PCR (UID assignment PCR cycles, see FIG. 2). While both forward and
reverse gene-specific primers contained universal tag sequences at
their 5' ends--providing the primer binding sites for the
second-round amplification--only the forward primer contained the
UID, positioned between the 5' universal tag and the 3'
gene-specific sequences (four N's were included in the reverse
primer to facilitate sequencing done on paired-end libraries)
(table S4). The UID assignment PCR cycles included Phusion Hot
Start II (Thermo Scientific) in a 50 .mu.L reaction containing
1.times. Phusion HF buffer, 0.25 mM dNTPs, 0.5 .mu.M each of
forward (containing 14 N's) and reverse primers, and 2 U of
polymerase. Carryover of residual UID-containing primers to the
second-round amplification, which can complicate template
quantification (30), was minimized through exonuclease digestion at
370 C to degrade unincorporated primers and subsequent purification
with AMPure XP beads (Beckman) and elution in 10 .mu.L TE (10 mM
Tris-HCl, 1 mM EDTA, pH 8.0).
[0080] The eluted templates were amplified in a second-round PCR
using primers containing the grafting sequences necessary for
hybridization to the Illumina GA IIx flow cell at their 5' ends
(FIG. 2) and two terminal 3' phosphorothioates to protect them from
residual exonuclease activity (30). The reverse amplification
primer additionally contained an index sequence between the 5
`grafting and 3` universal tag sequences to enable the PCR products
from multiple individuals to be simultaneously analyzed in the same
flow cell compartment of the sequencer (30). The second-round
amplification reactions contained 1.times. Phusion HF buffer, 0.25
mM dNTPs, 0.5 .mu.M each of forward and reverse primers, and 2 U of
polymerase in a total of 50 .mu.L. After an initial heat activation
step at 980 C for 2 minutes, twenty-three cycles of PCR were
performed using the following cycling conditions: 980 C for 10 s,
650 C for 15 s, and 720 C for 15 s. The multiplexed assay was
performed in similar fashion utilizing six independent
amplifications per sample with the primers described in table S5.
The PCR products were purified using AMPure XP beads and used
directly for sequencing on either the Illumina MiSeq or GA IIx
instruments, with equivalent results.
[0081] Data Analysis. High quality sequence reads were analyzed as
previously described. (30) Briefly, reads in which each of the 14
bases comprising the UID (representing one original template
strand; see FIG. 2) had a quality score .gtoreq.15 were grouped by
their UID. Only the UIDs supported by more than one read were
retained for further analysis. The template-specific portion of the
reads that contained the sequence of an expected amplification
primer was matched to a reference sequence set using a custom
script (available from the authors upon request). Artifactual
mutations--introduced during the sample preparation and/or
sequencing steps--were eliminated by requiring that >50% of
reads sharing the same UID contained the identical mutation (a
"supermutant;" see FIG. 2). For the 46 assays querying a single
amplicon, we required that the fraction of mutant alleles was
significantly different from the background mutation levels
determined from a negative control (P<0.001, binomial test). As
mutations are not known a priori in a screening environment, we
used a more agnostic metric to detect mutations in the multiplexed
assay. A threshold supermutant frequency was defined for each
sample as equaling the mean frequency of all supermutants plus six
standard deviations of the mean. Only supermutants exceeding this
threshold were designated as mutations and reported in FIG. 4 and
table S6.
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Sequence CWU 1
1
191165DNAArtificial Sequencecapture sequence 1agatcggaag agcacacgtc
tgaactccag tcacnnnnnn atctcgtatg ccgtcttctg 60cttgt
65215DNAArtificial SequencePrimers 2tgatcccgcg acgac
15315DNAArtificial SequencePrimers 3gaccgcgact ccagc
15460DNAArtificial SequencePrimers 4cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnngatcc aatccatttt tgttgtccag 60545DNAArtificial
SequencePrimers 5cacacaggaa acagctatga ccatgtgagc aagaggcttt ggagt
45652DNAArtificial SequencePrimers 6cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnnggcca agacctgccc tg 52747DNAArtificial
SequencePrimers 7cacacaggaa acagctatga ccatgtgctg tgactgcttg
tagatgg 47856DNAArtificial SequencePrimers 8cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnnccacc tcctcaaaca gctcaa 56946DNAArtificial
SequencePrimers 9cacacaggaa acagctatga ccatgtgcag cttgcttagg tccact
461055DNAArtificial SequencePrimers 10cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnntggcc atctacaagc agtca 551149DNAArtificial
SequencePrimers 11cacacaggaa acagctatga ccatgnnnnt caccatcgct
atctgagca 491255DNAArtificial SequencePrimers 12cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnncattg gtgatgattc gatgg
551345DNAArtificial SequencePrimers 13cacacaggaa acagctatga
ccatgctgcc tggctcagaa ttcac 451454DNAArtificial SequencePrimers
14cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccctt tcttgcggag attc
541545DNAArtificial SequencePrimers 15cacacaggaa acagctatga
ccatgctact gggacggaac agctt 451655DNAArtificial SequencePrimers
16cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggaag agaatctccg caaga
551745DNAArtificial SequencePrimers 17cacacaggaa acagctatga
ccatggcttc ttgtcctgct tgctt 451856DNAArtificial SequencePrimers
18cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggcct gtctcaatat cccaaa
561948DNAArtificial SequencePrimers 19cacacaggaa acagctatga
ccatgttgtt tttctgtttc tccctctg 482054DNAArtificial SequencePrimers
20cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncaagg cactcttgcc tacg
542154DNAArtificial SequencePrimers 21cacacaggaa acagctatga
ccatgcattt tcattatttt tattataagg cctg 542255DNAArtificial
SequencePrimers 22cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnctgtg
gtagtggcac cagaa 552349DNAArtificial SequencePrimers 23cacacaggaa
acagctatga ccatgnnnna agcggctgtt agtcactgg 492454DNAArtificial
SequencePrimers 24cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggccc
ctgtcatctt ctgt 542545DNAArtificial SequencePrimers 25cacacaggaa
acagctatga ccatggactt ggctgtccca gaatg 452658DNAArtificial
SequencePrimers 26cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncaaga
aatcgatagc atttgcag 582752DNAArtificial SequencePrimers
27cacacaggaa acagctatga ccatgtttat ttgctttgtc aagatcattt tt
522860DNAArtificial SequencePrimers 28cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnnaggaa atatctgctt gctcattcaa 602954DNAArtificial
SequencePrimers 29cacacaggaa acagctatga ccatggaagc agatactaag
caggacacta tatc 543055DNAArtificial SequencePrimers 30cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnnttccc ttggattctg acaca
553145DNAArtificial SequencePrimers 31cacacaggaa acagctatga
ccatgagcac cattcgttga taggc 453257DNAArtificial SequencePrimers
32cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncactg gcagcaacag tcttacc
573349DNAArtificial SequencePrimers 33cacacaggaa acagctatga
ccatggattg cctttaccac tcagagaag 493457DNAArtificial SequencePrimers
34cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnttgca gcaattcact gtaaagc
573555DNAArtificial SequencePrimers 35cacacaggaa acagctatga
ccatgccgat gtaataaata tgcacatatc attac 553654DNAArtificial
SequencePrimers 36cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncaagg
cactcttgcc tacg 543754DNAArtificial SequencePrimers 37cacacaggaa
acagctatga ccatgcattt tcattatttt tattataagg cctg
543857DNAArtificial SequencePrimers 38cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnncactg gcagcaacag tcttacc 573949DNAArtificial
SequencePrimers 39cacacaggaa acagctatga ccatggattg cctttaccac
tcagagaag 494054DNAArtificial SequencePrimers 40cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnncaagg cactcttgcc tacg
544154DNAArtificial SequencePrimers 41cacacaggaa acagctatga
ccatgcattt tcattatttt tattataagg cctg 544258DNAArtificial
SequencePrimers 42cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnagctc
aaagcaattt ctacacga 584353DNAArtificial SequencePrimers
43cacacaggaa acagctatga ccatgnnnng cacttacctg tgactccata gaa
534460DNAArtificial SequencePrimers 44cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnntcttt tgatgacatt gcatacattc 604549DNAArtificial
SequencePrimers 45cacacaggaa acagctatga ccatgnnnna ctccaaagcc
tcttgctca 494655DNAArtificial SequencePrimers 46cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnncagtt gcaaaccaga cctca
554749DNAArtificial SequencePrimers 47cacacaggaa acagctatga
ccatgtgtgg agtatttgga tgacagaaa 494853DNAArtificial SequencePrimers
48cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngtggc aagtggctcc tga
534945DNAArtificial SequencePrimers 49cacacaggaa acagctatga
ccatgnnnnc atgggcggca tgaac 455057DNAArtificial SequencePrimers
50cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnactgg cagcaacagt cttacct
575149DNAArtificial SequencePrimers 51cacacaggaa acagctatga
ccatgnnnnc ctcaggattg cctttacca 495253DNAArtificial SequencePrimers
52cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnagtcc ggcttggagg atg
535345DNAArtificial SequencePrimers 53cacacaggaa acagctatga
ccatgtcccc actcctcctt tcttc 455454DNAArtificial SequencePrimers
54cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggaaa gggacgaact ggtg
545549DNAArtificial SequencePrimers 55cacacaggaa acagctatga
ccatgnnnnt agggcctctt gtgccttta 495655DNAArtificial SequencePrimers
56cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntcttc tgtcccttcc cagaa
555749DNAArtificial SequencePrimers 57cacacaggaa acagctatga
ccatgnnnng acttggctgt cccagaatg 495858DNAArtificial SequencePrimers
58cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntcata ccaatttctc gattgagg
585949DNAArtificial SequencePrimers 59cacacaggaa acagctatga
ccatgnnnnc ggctttttca acccttttt 496055DNAArtificial SequencePrimers
60cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggcc atctacaagc agtca
556149DNAArtificial SequencePrimers 61cacacaggaa acagctatga
ccatgnnnnt caccatcgct atctgagca 496254DNAArtificial SequencePrimers
62cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngggac ggaacagctt tgag
546350DNAArtificial SequencePrimers 63cacacaggaa acagctatga
ccatgnnnng cggagattct cttcctctgt 506454DNAArtificial
SequencePrimers 64cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncggtg
taggagctgc tggt 546548DNAArtificial SequencePrimers 65cacacaggaa
acagctatga ccatgnnnna cccaggtcca gatgaagc 486655DNAArtificial
SequencePrimers 66cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggcc
atctacaagc agtca 556749DNAArtificial SequencePrimers 67cacacaggaa
acagctatga ccatgnnnnt caccatcgct atctgagca 496853DNAArtificial
SequencePrimers 68cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngtggc
aagtggctcc tga 536945DNAArtificial SequencePrimers 69cacacaggaa
acagctatga ccatgnnnnc atgggcggca tgaac 457057DNAArtificial
SequencePrimers 70cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncgaaa
agtgtttctg tcatcca 577149DNAArtificial SequencePrimers 71cacacaggaa
acagctatga ccatgnnnng cccctcctca gcatcttat 497255DNAArtificial
SequencePrimers 72cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncagtt
gcaaaccaga cctca 557353DNAArtificial SequencePrimers 73cacacaggaa
acagctatga ccatgnnnnt gtggagtatt tggatgacag aaa 537456DNAArtificial
SequencePrimers 74cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnttata
tttccccatg ccaatg 567559DNAArtificial SequencePrimers 75cacacaggaa
acagctatga ccatgnnnng gtgttttgaa atgtgtttta taatttaga
597655DNAArtificial SequencePrimers 76cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnntcttc tgtcccttcc cagaa 557749DNAArtificial
SequencePrimers 77cacacaggaa acagctatga ccatgnnnng acttggctgt
cccagaatg 497855DNAArtificial SequencePrimers 78cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnngaaaa agccgaaggt cacaa
557951DNAArtificial SequencePrimers 79cacacaggaa acagctatga
ccatgnnnnc tcaagaagca gaaagggaag a 518060DNAArtificial
SequencePrimers 80cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngatcc
aatccatttt tgttgtccag 608149DNAArtificial SequencePrimers
81cacacaggaa acagctatga ccatgnnnnt gagcaagagg ctttggagt
498255DNAArtificial SequencePrimers 82cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnntgtga tgatggtgag gatgg 558357DNAArtificial
SequencePrimers 83cacacaggaa acagctatga ccatgnnnnt ccactacaac
tacatgtgta acagttc 578455DNAArtificial SequencePrimers 84cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnnctccg tcatgtgctg tgact
558548DNAArtificial SequencePrimers 85cacacaggaa acagctatga
ccatgnnnnc agctgtgggt tgattcca 488655DNAArtificial SequencePrimers
86cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggcc atctacaagc agtca
558749DNAArtificial SequencePrimers 87cacacaggaa acagctatga
ccatgnnnnt caccatcgct atctgagca 498856DNAArtificial SequencePrimers
88cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccaat ccatttttgt tgtcca
568949DNAArtificial SequencePrimers 89cacacaggaa acagctatga
ccatgnnnnt gagcaagagg ctttggagt 499053DNAArtificial SequencePrimers
90cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnctgca cagggcaggt ctt
539153DNAArtificial SequencePrimers 91cacacaggaa acagctatga
ccatgnnnnc tctgtctcct tcctcttcct aca 539255DNAArtificial
SequencePrimers 92cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggcc
atctacaagc agtca 559349DNAArtificial SequencePrimers 93cacacaggaa
acagctatga ccatgnnnnt caccatcgct atctgagca 499455DNAArtificial
SequencePrimers 94cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncacgc
aaatttcctt ccact 559550DNAArtificial SequencePrimers 95cacacaggaa
acagctatga ccatgnnnng gcctctgatt cctcactgat 509660DNAArtificial
SequencePrimers 96cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngatcc
aatccatttt tgttgtccag 609749DNAArtificial SequencePrimers
97cacacaggaa acagctatga ccatgnnnnt gagcaagagg ctttggagt
499857DNAArtificial SequencePrimers 98cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnnactgg cagcaacagt cttacct 579949DNAArtificial
SequencePrimers 99cacacaggaa acagctatga ccatgnnnnc ctcaggattg
cctttacca 4910062DNAArtificial SequencePrimers 100gacgtaaaac
gacggccagt nnnnnnnnnn nnnnaaagta acatttccaa tctactaatg 60ct
6210150DNAArtificial SequencePrimers 101cacacaggaa acagctatga
ccatgnnnnt gagaaaatcc ctgttcccac 5010255DNAArtificial
SequencePrimers 102cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntggag
cctcttacac ccagt 5510351DNAArtificial SequencePrimers 103cacacaggaa
acagctatga ccatgnnnna aaaacactgg agtttcccaa a 5110455DNAArtificial
SequencePrimers 104cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngaaaa
agccgaaggt cacaa 5510549DNAArtificial SequencePrimers 105cacacaggaa
acagctatga ccatgnnnna tgcccccaag aatcctagt 4910655DNAArtificial
SequencePrimers 106cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncatcc
gtctactccc acgtt 5510748DNAArtificial SequencePrimers 107cacacaggaa
acagctatga ccatgnnnna tcagctaccg ccaagtcc 4810855DNAArtificial
SequencePrimers 108cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnttcgt
ccctttccag cttta 5510956DNAArtificial SequencePrimers 109cacacaggaa
acagctatga ccatgnnnng gaatccagtg tttcttttaa atacct
5611054DNAArtificial SequencePrimers 110cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnnaccag ccctgtcgtc tctc 5411151DNAArtificial
SequencePrimers 111cacacaggaa acagctatga ccatgnnnng ccctgacttt
caactctgtc t 5111259DNAArtificial SequencePrimers 112cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnngcctc agattcactt ttatcacct
5911349DNAArtificial SequencePrimers 113cacacaggaa acagctatga
ccatgnnnna ccaggagcca ttgtctttg 4911460DNAArtificial
SequencePrimers 114cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntcacc
acattacata cttaccatgc 6011551DNAArtificial SequencePrimers
115cacacaggaa acagctatga ccatgnnnna aggggatctc ttcctgtatc c
5111655DNAArtificial SequencePrimers 116cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnntctgg atcccagaag gtgag 5511749DNAArtificial
SequencePrimers 117cacacaggaa acagctatga ccatgnnnng gccagtgctg
tctctaagg 4911860DNAArtificial SequencePrimers 118cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnngtcca caaaatgatt ctgaattagc
6011951DNAArtificial SequencePrimers 119cacacaggaa acagctatga
ccatgnnnna cgatacacgt ctgcagtcaa c 5112062DNAArtificial
SequencePrimers 120cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnacaga
aatattttag aaagggacaa 60ca 6212151DNAArtificial SequencePrimers
121cacacaggaa acagctatga ccatgnnnna gaaaataccc cctccatcaa c
5112260DNAArtificial SequencePrimers 122cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnngatcc aatccatttt tgttgtccag 6012353DNAArtificial
SequencePrimers 123cacacaggaa acagctatga ccatgnnnnt ccaaactgac
caaactgttc tta 5312457DNAArtificial SequencePrimers 124cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnngaaac ccaaaatctg ttttcca
5712550DNAArtificial SequencePrimers 125cacacaggaa acagctatga
ccatgnnnng accataaccc accacagcta 5012655DNAArtificial
SequencePrimers 126cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnctcct
cccagagacc ccagt 5512749DNAArtificial SequencePrimers 127cacacaggaa
acagctatga ccatgnnnnc atgagcgctg ctcagatag 4912854DNAArtificial
SequencePrimers 128cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncctag
gaaggcaggg gagt 5412949DNAArtificial SequencePrimers 129cacacaggaa
acagctatga ccatgnnnnt gcatgttgct tttgtaccg 4913054DNAArtificial
SequencePrimers 130cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnaccac
ccgcacgtct gtag 5413148DNAArtificial SequencePrimers 131cacacaggaa
acagctatga ccatgnnnna gccagtgctt gttgcttg 4813254DNAArtificial
SequencePrimers 132cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncacac
tgacgtgcct ctcc 5413349DNAArtificial SequencePrimers 133cacacaggaa
acagctatga ccatgnnnnt tatctcccct ccccgtatc 4913455DNAArtificial
SequencePrimers 134cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngattg
tcagtgcgct tttcc 5513549DNAArtificial SequencePrimers 135cacacaggaa
acagctatga ccatgnnnng ctaaggatgg gggttgcta 4913661DNAArtificial
SequencePrimers 136cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgact
ttaccttatc aatgtctcga 60a 6113748DNAArtificial SequencePrimers
137cacacaggaa acagctatga ccatgnnnng ctcgccccct taatctct
4813855DNAArtificial SequencePrimers 138cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnnggtac ttccggaacc tgtgc 5513949DNAArtificial
SequencePrimers 139cacacaggaa acagctatga ccatgnnnnc cgagtcctag
ggagaggag 4914058DNAArtificial SequencePrimers 140cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnnttgtt aatggtggct ttttgttt
5814154DNAArtificial SequencePrimers 141cacacaggaa acagctatga
ccatgnnnna aatgatctaa caatgctctt ggac 5414258DNAArtificial
SequencePrimers 142cgacgtaaaa cgacggccag tnnnnnnnnn nnnnncatgg
aaggatgaga atttcaag 5814349DNAArtificial SequencePrimers
143cacacaggaa acagctatga ccatgnnnnt ggctacgacc cagttacca
4914455DNAArtificial SequencePrimers 144cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnnaaacc gtagctgccc tggta 5514550DNAArtificial
SequencePrimers 145cacacaggaa acagctatga ccatgnnnnt gactgctctt
ttcacccatc 5014656DNAArtificial SequencePrimers 146cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnntcatc ttgggcctgt gttatc
5614753DNAArtificial SequencePrimers 147cacacaggaa acagctatga
ccatgnnnng atgagaggtg gatgggtagt agt 5314856DNAArtificial
SequencePrimers 148cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnttcag
ggcatgaact acttgg 5614949DNAArtificial SequencePrimers
149cacacaggaa acagctatga ccatgnnnna tcctcccctg catgtgtta
4915056DNAArtificial SequencePrimers 150cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnntccct cattgcactg tactcc 5615149DNAArtificial
SequencePrimers 151cacacaggaa acagctatga ccatgnnnng gtgcttagtg
gccatttgt 4915257DNAArtificial SequencePrimers 152cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnntggtc tctcatggca ctgtact
5715352DNAArtificial SequencePrimers 153cacacaggaa acagctatga
ccatgnnnna ttagcaattt gagggacaaa cc 5215462DNAArtificial
SequencePrimers 154cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgtat
ctcactcgat aatctggatg 60ac 6215555DNAArtificial SequencePrimers
155cacacaggaa acagctatga ccatgnnnnt gtcacattat aaagattcag gcaat
5515659DNAArtificial SequencePrimers 156cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnnagttt gacagttaaa ggcatttcc 5915754DNAArtificial
SequencePrimers 157cacacaggaa acagctatga ccatgnnnnt gtccttattt
tggatatttc tccc 5415855DNAArtificial SequencePrimers 158cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnngaaga cccaggtcca gatga
5515948DNAArtificial SequencePrimers 159cacacaggaa acagctatga
ccatgnnnna gaaatgcagg gggatacg 4816055DNAArtificial SequencePrimers
160cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngaggc ataactgcac ccttg
5516149DNAArtificial SequencePrimers 161cacacaggaa acagctatga
ccatgnnnng ggagtagatg gagcctggt 4916255DNAArtificial
SequencePrimers 162cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgctg
gatttggttc taggg 5516350DNAArtificial SequencePrimers 163cacacaggaa
acagctatga ccatgnnnnt gccacttgca aagtttcttc 5016455DNAArtificial
SequencePrimers 164cgacgtaaaa cgacggccag tnnnnnnnnn
nnnnnggaag aacctggacc ctctg 5516550DNAArtificial SequencePrimers
165cacacaggaa acagctatga ccatgnnnnt tttgagagtc gttcgattgc
5016655DNAArtificial SequencePrimers 166cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnntgcaa cctgttttgt gatgg 5516751DNAArtificial
SequencePrimers 167cacacaggaa acagctatga ccatgnnnna ggaaaatgac
aatgggaatg a 5116859DNAArtificial SequencePrimers 168cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnntgatt catcaggaga gcatttaag
5916952DNAArtificial SequencePrimers 169cacacaggaa acagctatga
ccatgnnnnt tgtttttctg tttctccctc tg 5217057DNAArtificial
SequencePrimers 170cgacgtaaaa cgacggccag tnnnnnnnnn nnnnngatgg
tatccatgtg gtgagtg 5717150DNAArtificial SequencePrimers
171cacacaggaa acagctatga ccatgnnnnt ttgtgatgct aaggctccat
5017253DNAArtificial SequencePrimers 172cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnnactgc cttccgggtc act 5317349DNAArtificial
SequencePrimers 173cacacaggaa acagctatga ccatgnnnna gcccaaccct
tgtccttac 4917454DNAArtificial SequencePrimers 174cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnngaggc tgtcagtggg gaac
5417550DNAArtificial SequencePrimers 175cacacaggaa acagctatga
ccatgnnnna acatatttgc atggggtgtg 5017655DNAArtificial
SequencePrimers 176cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccact
gcatggttca ctctg 5517749DNAArtificial SequencePrimers 177cacacaggaa
acagctatga ccatgnnnna tcctgtgagc gaagttcca 4917857DNAArtificial
SequencePrimers 178cgacgtaaaa cgacggccag tnnnnnnnnn nnnnntgcct
ctttctcttg gttttca 5717951DNAArtificial SequencePrimers
179cacacaggaa acagctatga ccatgnnnng gacctaagca agctgcagta a
5118060DNAArtificial SequencePrimers 180cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnnacacc caatgaagaa tgtaattgat 6018154DNAArtificial
SequencePrimers 181cacacaggaa acagctatga ccatgnnnng gttgtgtgta
gatgtgagtt ttcc 5418259DNAArtificial SequencePrimers 182cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnnttctg ttacattgtg cagagttca
5918350DNAArtificial SequencePrimers 183cacacaggaa acagctatga
ccatgnnnnt ggttttgagc agagagatgg 5018455DNAArtificial
SequencePrimers 184cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnggaag
aagtcccaac catga 5518553DNAArtificial SequencePrimers 185cacacaggaa
acagctatga ccatgnnnnt cacttttcct ttctacccaa aag
5318656DNAArtificial SequencePrimers 186cgacgtaaaa cgacggccag
tnnnnnnnnn nnnnngaatc tgcattccca gagaca 5618749DNAArtificial
SequencePrimers 187cacacaggaa acagctatga ccatgnnnnc ctgtttccca
tcctcttcc 4918856DNAArtificial SequencePrimers 188cgacgtaaaa
cgacggccag tnnnnnnnnn nnnnngacac aaaacaggct caggac
5618949DNAArtificial SequencePrimers 189cacacaggaa acagctatga
ccatgnnnna gaaaccaaag ccaaaagca 4919055DNAArtificial
SequencePrimers 190cgacgtaaaa cgacggccag tnnnnnnnnn nnnnnccatg
ggactgactt tctgc 5519149DNAArtificial SequencePrimers 191cacacaggaa
acagctatga ccatgnnnnt catctggacc tgggtcttc 49
* * * * *
References